OBJECTIVETo establish a specific TaqMan-based Real-time PCR assay for the detection of hepatitis E virus (HEV).

METHODSAccording to the references, primers-probe sets which were located in ORF2, the conservative part of HEV genome were designed and therefore we established a HEV TaqMan real-time RT-PCR assay with great performance of specificity, sensitivity and reproducibility. And then it was used in the detection of HEV RNA in clinical samples.

RESULTSThe HEV Real-time RT-PCR assay established in this study were able to detect HEV RNA with a detection limit of 10 copies/reaction. When the detection of a same sample was repeated for several times, coefficients of variation (CV) was all less than 1.53%. Our data also suggested that there were 1.87 x 10(6)-8.12 x 10(9) RNA copies in 1 ml of the clinical samples.

CONCLUSIONThe TaqMan-based Real-time PCR assay established in this study was specific and precise for the rapid detection of HEV RNA. It was applied successfully in the pathogen detection of clinical samples.

DNA Primers ; genetics ; Hepatitis E ; virology ; Hepatitis E virus ; genetics ; isolation & purification ; Humans ; RNA, Viral ; genetics ; metabolism ; Real-Time Polymerase Chain Reaction ; methods ; Taq Polymerase ; metabolism

OBJECTIVETo investigate the effect of miR-122 on the expression of hepatitis B virus (HBV) proteins.

METHODSAnti-sense oligodeoxynucleotide (ASODN) of two different sequences against miR-122, anti-miR-122 and LNA-antimiR-122 (Locked nucleic acid), human miR -122 (hsa-miR-122), or the negative control anti-GFP were designed and synthesized, then transfected into HepG2.2.15 cells. After 24 h and 48 h, the levels of HBsAg and HBeAg in the supernatant were determined with a time-resolved immunofluorometric assay (TRFIA). HBV DNA in supernatant and miR-122 in cells were measured by quantitative real-time PCR.

RESULTSAfter 48 h expressions of miR-122 in the LNA-antimiR-122 and anti-miR-122 groups were significantly suppressed and lower than those in the negative control (P<0.001), while the level of miR-122 in the hsa-miR-122 group was higher than that in the negative control (P<0.001). The expression of HBeAg and HBsAg in hsa-miR-122 group was lower than that in the negative control (P<0.01) 24 h and 48 h after transfection. The expression of HBeAg and HBsAg in the anti-miR-122 group and LNA-antimiR-122 group was significantly lower than that in negative control (P>0.001). The levels of viral DNA at both time-points in the various test groups were not significantly different from those of negative control group (P>0.05).

CONCLUSIONmiR-122 may regulate HBV antigens and potentially affect the progress of pathogenesis, which might be the new targets for treatment of HBV infection.

DNA, Viral ; genetics ; Hep G2 Cells ; Hepatitis B Surface Antigens ; metabolism ; Hepatitis B e Antigens ; metabolism ; Hepatitis B virus ; genetics ; Humans ; MicroRNAs ; genetics ; metabolism ; Transfection

Replication of the hepatitis B virus is suppressed by deficiency of the X protein. Although several molecules that block cellular targets of X protein reduce the production of hepatitis B virus progeny, the effect of a specific inhibitor of X protein on viral replication has not been investigated. To block X protein specifically, we adopted an intracellular expression approach using H7 single chain variable fragment (H7scFv), an antibody fragment against X protein. We previously demonstrated that cytoplasmic expression of H7scFv inhibits X protein-induced tumorigenicity and transactivation. In this study, intracellular H7scFv expression inhibits reporter gene transactivation but not viral replication determined by endogenous hepatitis B virus polymerase activity assay and real-time PCR. Our findings imply that intracellular expression of antibody fragment against X protein may not be an alternative therapeutic modality for inhibition of hepatitis B virus replication.
Virus Replication/*drug effects ; Trans-Activators/*antagonists & inhibitors/immunology ; Immunoglobulin Variable Region/genetics/metabolism/*pharmacology ; Hepatitis B virus/*drug effects/physiology ; Hepatitis B e Antigens/metabolism ; Cell Line

OBJECTIVETo investigate the effect of apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3G (APOBEC3G) mediated antiviral activity against hepatitis B virus (HBV) and duck hepatitis B virus (DHBV).

METHODSTotal RNA was extracted from peripheral blood mononuclear cells (PBMCs), RT-PCR product was cloned into the EcoR I/Hind III restriction sites of the CMV-driven expression vector fused with a hemagglutinin fusion epitope tag at its carboxyl terminal. Replication competent 1.3 fold over-length HBV was constructed with full-length HBV of ayw subtype. The mammalian hepatoma cell HepG2 was cotransfected with the replication competent 1.3 fold over-length HBV and various amounts of CMV-driven expression vector encoding APOBEC3G-HA. Levels of HBsAg and HBeAg in the media of the transfected cells were determined by ELISA, HBV DNA. RNA from intracellular core particles was examined using Northern and Southern blot analyses. Chicken hepatoma cell LMH was cotransfected with head-to-tail dimer of an EcoR I monomer of DHBV and various amounts of CMV-driven expression vector encoding APOBEC3G-HA. DHBV DNA from intracellular core particles was examined using Southern blot analysis.

RESULTSCMV-driven expression vector encoding APOBEC3G-HA and replication competent 1.3 fold over-length HBV were constructed. There was a dose dependent decrease in the levels of intracellular core-associated viral (HBV and DHBV) DNA and extracellular production of HBsAg and HBeAg. Levels of intracellular core-associated viral RNA were also decreased, but the expression of HBcAg remained almost unchanged.

CONCLUSIONAPOBEC3G suppresses HBV and DHBV replication and also suppresses HBsAg and HBeAg expression.

APOBEC-3G Deaminase ; Cytidine Deaminase ; genetics ; Hep G2 Cells ; Hepatitis B Surface Antigens ; metabolism ; Hepatitis B Virus, Duck ; physiology ; Hepatitis B e Antigens ; metabolism ; Hepatitis B virus ; physiology ; Humans ; RNA, Messenger ; genetics ; Virus Replication

OBJECTIVETo investigate the roles of the hepatitis B virus (HBV)-encoded X protein (HBx), including the full-length and truncated isoforms, and in conjunction with the host-encoded damaged DNA binding protein 1 (DDB1) in HBV replication.

METHODSRecominant expression plasmids carrying the wild-type HBV genome (pGEM-HBV1.2) or with deletion of the full-length HBx protein (pHBV-deltaX), or carrying the full-length HBx protein (pSI-X) or the HBx1to101 (pSI-X1to101) or HBx43to154 (pSI-X43to154) isoforms were constructed for transfection into HepG2 cells. The pcDNA6.2-GW/EmGFP-miR (DDB1-miRNA) vector was constructed for silencing of the DDB1 gene in co-transfected HepG2 cells. At 72 h after transfections, DDB1 silencing was confirmed by western blot analysis and real-time quantitive reverse transcription PCR, HBV DNA copies number was assessed by real time PCR, and levels of hepatitis B surface antigen (HbsAg) and hepatitis B e antigen (HbeAg) were determined by ELISA. Differences between groups was statistically analyzed by single-factor analysis of variance and the t-test.

RESULTSTransfection with pHBV-deltaX led to reductions in DDB1 mRNA (to 52.74% of that in the wild-type pGEM-HBV1.2 transfected cells), HBV replication (to 55.49%), HBsAg level (48.05%), and HBeAg level (46.22%). Co-transfection with pSI-X or pSI-X43to154, but not with pSI-X1to101, restored the pHBV-deltaX-induced reductions in DDB1 mRNA, HBV replication, HBsAg and HBeAg to wild-type levels. The quantity of DDB1 mRNA was approximately parallel with the quantity of HBV DNA copies in all the HepG2 transfection groups.

CONCLUSIONThe COOH-terminal amino acids of HBx are required for HBV replication in hepatocytes, possibly involving the host-encoded DDB1 protein.

DNA-Binding Proteins ; metabolism ; Hep G2 Cells ; Hepatitis B Surface Antigens ; metabolism ; Hepatitis B e Antigens ; metabolism ; Hepatitis B virus ; metabolism ; physiology ; Host-Pathogen Interactions ; Humans ; Protein Isoforms ; metabolism ; Trans-Activators ; metabolism ; Transfection ; Virus Replication

OBJECTIVETo evaluate the effect of heat shock protein 90 (HSP90) on hepatitis B virus (HBV) replication in hepatocytes and to investigate the related molecular mechanism.

METHODSA eukaryotic plasmid expressing human HSP90 was constructed (designated as HA-HSP90). HepG2 cells were co-transfected with HA-HSP90 and the HBV replicative plasmid HBV1.3. Expression of the exogenous HSP90 was assessed by Western blotting. Expression of the HBV surface antigen (HBsAg) was determined by enzyme-linked immunosorbent assay, and HBV replicative intermediates were detected by Southern blotting. Small interfering (si)RNAs were designed against HSP90 and TBK1 and transfected into the HepG2 cells to further assess the effects of HSP90 and its underlying mechanism. HSP90-mediated effects on the expression of interleukins IL-1b and IL-6 and the interferon response gene IFIT1 were assessed by quantitating mRNA levels with real time RT-PCR.

RESULTSThe HA-HSP90 plasmid successfully expressed exogenous HSP90 protein in HepG2 cells. The exogenous HSP90 was able to inhibit HBV replication and HBsAg expression. IFIT1 expression was up-regulated after HA-HSP90 transfection, but neither IL-1b nor IL-6 were affected. The siRNA-mediated TBK1 down-regulation had no effect on the HSP90-inhibited HBV replication.

CONCLUSIONHSP90 can inhibit HBV replication and TBK1 is not involved in this process.

HSP90 Heat-Shock Proteins ; genetics ; Hep G2 Cells ; Hepatitis B e Antigens ; metabolism ; Hepatitis B virus ; physiology ; Humans ; Protein-Serine-Threonine Kinases ; genetics ; Transfection ; Virus Replication

OBJECTIVEo study the replication of hepatitis B virus (HBV) in HepG2 cells infected with Ad-1.2 HBV.

METHODSHepG2 cells were transfected with adenovirus containing 1.2 copies of HBV DNA. The expression of HBV antigens were detected in the culture medium by means of enzyme-linked immunosorbent assay (ELISA), and the covalently closed circular DNA (cccDNA) in the cells was extracted with plasmid extraction kit and detected by real-time PCR with selective primer after treatment with mung bean nuclease.

RESULTSHBsAg, HBeAg and HBV cccDNA were all detected in HepG2 cells after tranfection with Ad-1.2 HBV. HBV cccDNA was detected 1 day after the infection, reaching the peak level 4 days after infection.

CONCLUSIONAd-1.2 HBV-infected cells can serve as the model for screening and evaluation of antiviral agents.

Adenoviridae ; genetics ; Calibration ; Cell Line, Tumor ; DNA, Complementary ; genetics ; metabolism ; DNA, Viral ; genetics ; metabolism ; Hepatitis B Surface Antigens ; metabolism ; Hepatitis B e Antigens ; metabolism ; Hepatitis B virus ; genetics ; immunology ; metabolism ; physiology ; Humans ; Polymerase Chain Reaction ; Time Factors ; Transfection ; Virus Replication

OBJECTIVETo screen proteins in hepatocytes interacting with HBeAg transactivated protein (HBeAgTP) with yeast-two hybrid technique for investigating the biological functions of HBeAgTP.

METHODSSuppression subtractive hybridization (SSH) and bioinformatics techniques were used for screening and cloning of the target genes transactivated by HBeAg. The HBeAgTP gene was amplified by polymerase chain reaction (PCR) and HBeAgTP bait plasmid was constructed with yeast-two hybrid system 3, and then transformed into yeast AH109. The transformed yeast mated with yeast Y187 containing liver cDNA library plasmid in 2 x YPDA medium. Diploid yeast was plated on synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) and synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) containing X-gal for selecting two times and screening. After extracting and sequencing of plasmid from blue colonies, the results were analyzed by bioinformatics.

RESULTSHBeAgTP gene was successfully cloned and expressed in yeast cells. Fifteen genes in twenty-four positive colonies were obtained using yeast-two hybrid technique.

CONCLUSIONHBeAgTP conjugated protein genes were successfully cloned, along with the genes involved in transcription and translation of proteins, immunoloregulation, materials and energy metabolism in vivo.

Hepatitis B e Antigens ; genetics ; metabolism ; Hepatitis B virus ; immunology ; Hepatocytes ; immunology ; metabolism ; Humans ; Protein Interaction Mapping ; Protein Precursors ; genetics ; metabolism ; Two-Hybrid System Techniques ; Yeasts ; genetics

BACKGROUNDTo determine the antigenicity of recombinant hepatitis E virus ORF2 (rHEV ORF2) protein expressed in Pichia pastoris (P. pastoris).

METHODSBy using the rHEV ORF2 protein from E.coli as control, an indirect ELISA was adopted to identify the sensitivity, specificity and stability of rHEV ORF2 protein from P. pastoris in detection of HEV IgM and IgG antibody in sera from patients with hepatitis E. The reactivity of the rHEV ORF2 against 5 HEV ORF2 monoclonal antibodies (McAbs) was also tested.

RESULTSThe minimum concentration of coated antigen with which HEV IgG could be detected was 12.5 ng/ml, while the highest serum dilution to detect both IgM and IgG antibodies against HEV was 1:5 120. No cross-reaction was found with sera from patients with any other types of hepatitis. The 37 degree C acceleration test showed that the rORF2 was highly stable within 12 months at 4 degrees C. The 5 HEV ORF2 McAbs showed better reaction with the rORF2 from P. pastoris, especially that 4B2, 2E2, whose reaction against the rORF2 were 125 and 25 times respectively higher than that of rORF2 from E.Coli.

CONCLUSIONThere may be more extensive conformational epitopes in the rHEV ORF2 from P. pastoris. The excellent antigenicity, sensitivity and stability suggest that it can be served as a new candidate antigen for the development of diagnostic reagents of hepatitis E.

Gene Expression ; Hepatitis Antibodies ; blood ; Hepatitis Antigens ; genetics ; immunology ; Hepatitis E ; immunology ; Hepatitis E virus ; genetics ; immunology ; Humans ; Pichia ; genetics ; metabolism ; Recombinant Proteins ; genetics ; immunology ; Viral Proteins ; genetics ; immunology

OBJECTIVESTo evaluate the inhibitory effect mediated by combination of small interfering RNAs (siRNAs) targeting different sites of hepatitis B virus (HBV) transcripts on the viral replication and antigen expression in vitro.

METHODS(1) Seven siRNAs targeting surface (S), polymerase (P) or precore (PreC) region of HBV genome were designed and chemically synthesized. (2) HBV-producing HepG2.2.15 cells were treated with or without siRNAs for 72 h. (3) HBsAg and HBeAg in the cell culture medium were detected by enzyme-linked immunoadsorbent assay. (4) Intracellular viral DNA was quantified by real-time PCR (Polymerase Chain Reaction). (5) HBV viral mRNA was reverse transcribed and quantified by real-time PCR. (6) The change of cell cycle and apoptosis was determined by flow cytometry.

RESULTSOur data demonstrated that synthetic small interfering RNAs (siRNAs) targeting S and PreC gene could efficiently and specifically inhibit HBV replication and antigen expression. The expression of HBsAg and HBeAg and the replication of HBV could be specifically inhibited in a dose-dependent manner by siRNAs. Furthermore, our results showed that the combination of siRNAs targeting various regions could inhibit HBV replication and antigen expression in a more efficient way than the use of single siRNA at the same final concentration. No apoptotic change was observed in the cell after siRNA treatment.

CONCLUSIONOur results demonstrated that siRNAs exerted robust and specific inhibition on HBV replication and antigen expression in a cell culture system and combination of siRNAs targeting different regions exhibited more potency.

Apoptosis ; Cell Cycle ; Cell Line, Tumor ; DNA, Viral ; biosynthesis ; Flow Cytometry ; Gene Expression Regulation, Viral ; genetics ; Hepatitis B Surface Antigens ; metabolism ; Hepatitis B e Antigens ; metabolism ; Hepatitis B virus ; genetics ; physiology ; Humans ; RNA, Small Interfering ; genetics ; metabolism ; Virus Replication ; genetics